Answer to Question #256780 in Organic Chemistry for iyoss

Question

. A solution containing egg albumin (pI=4.6), β-lactoglobulin (pI=5.2), and chymotrypsinogen (pI=9.5) was loaded onto a column of diethylaminoethyl cellulose (DEAE-cellulose) at pH 5.4.The column was then eluted with a pH 5.4 buffer, with an increasing salt concentration. Predict the elution pattern. 

Answer 1

The protein purification is possible in an ion-exchange column based on their isoelectric poin (pI). The isoelectric point (pI) is the pH of a solution at which the net charge of a protein becomes zero. If the solution pH that is above the pI (pH >pI), the surface of the protein is predominantly negatively charged (an anion). If the solution pH is below the pI (pH <pI) the surface of the protein is predominantly positively charged ( a cation).

Here the pH of the solution is 5.4, the pI for egg albumin (pI=4.6), here pH > pI the protein is negatively charged, the pI for b-lactoglobulin (pI=5.2), here pH > pI the protein is negatively charged and pI for chymotrypsinogen (pI=9.5), here pH < pI the protein is positively charged.

Here we are using diethylaminoethyl cellulose (DEAE-Cellulose) which is a positively charged resin used in ion-exchange chromatography. So here are using an anion exchange resin the negative charge are retained in the column and the positive charge is first eluted because anion resins have a positive charge and positive charged protein repels. Then the separation is based on the electron density or charge density. Lower charge density will be eluted then and the resin will retain the protein with higher electron density. Here since pI of egg albumin is less than b-lactoglobulin, thus have greater negative charge density than b-lactoglobulin.

The order in which the proteins elute from the column:

1st - chymotrypsinogen

2nd -β-lactoglobulin

3rd - egg albumin